Multidimensional kinetic analysis of immobilized enzymes is essential to understand the enzyme functionality at the interface with solid materials. However, spatiotemporal kinetic characterization of heterogeneous biocatalysts on a microscopic level and under operando conditions has been rarely approached. As a case study, we selected self-sufficient heterogeneous biocatalysts where His-tagged cofactor-dependent enzymes (dehydrogenases, transaminases, and oxidases) are co-immobilized with their corresponding phosphorylated cofactors [nicotinamide adenine dinucleotide phosphate (NAD(P)H), pyridoxal phosphate (PLP), and flavin adenine dinucleotide (FAD)] on porous agarose microbeads coated with cationic polymers. These self-sufficient systems do not require the addition of exogenous cofactors to function, thus avoiding the extensive use of expensive cofactors. To comprehend the microscopic kinetics and thermodynamics of self-sufficient systems, we performed fluorescence recovery after photobleaching measurements, time-lapse fluorescence microscopy, and image analytics at both single-particle and intraparticle levels. These studies reveal a thermodynamic equilibrium that rules out the reversible interactions between the adsorbed phosphorylated cofactors and the polycations within the pores of the carriers, enabling the confined cofactors to access the active sites of the immobilized enzymes. Furthermore, this work unveils the relationship between the apparent Michaelis–Menten kinetic parameters and the enzyme density in the confined space, eliciting a negative effect of molecular crowding on the performance of some enzymes. Finally, we demonstrate that the intraparticle apparent enzyme kinetics are significantly affected by the enzyme spatial organization. Hence, multiscale characterization of immobilized enzymes serves as an instrumental tool to better understand the in operando functionality of enzymes within confined spaces.

Alcohol dehydrogenase from Bacillus (Geobacillus) stearothermophilus (BsADH) is a NADH-dependent enzyme catalyzing the oxidation of alcohols, however its thermal and operational stabilities are too low for its long-term use under non-physiological conditions. Enzyme immobilizations emerges as an attractive tool to enhance the stability of this enzyme. In this work, we have screened a battery of porous carriers and immobilization chemistries to enhance the robustness of a His-tagged variant of BsADH. The selected carriers recovered close to 50% of the immobilized activity and increased enzyme stability from 3 to 9 times compared to the free enzyme. We found a trade-off between the half-life time and the specific activity as a function of the relative anisotropy values of the immobilized enzymes, suggesting that both properties are oppositely related to the enzyme mobility (rotational tumbling). The most thermally stable heterogeneous biocatalysts were coupled with a NADH oxidase/catalase pair co-immobilized on porous agarose beads to perform the batch oxidation of five different 1,ω-diols with in situ recycling of NAD+. Only when His-tagged BsADH was immobilized on porous glass functionalized with Fe3+, the heterogeneous biocatalyst oxidized 1, 5-pentanediol with a conversion higher than 50% after five batch cycles. This immobilized multi-enzyme system presented promising enzymatic productivities towards the oxidation of three different diols. Hence, this strategical study accompanied by a functional and structural characterization of the resulting immobilized enzymes, allowed us selecting an optimal heterogeneous biocatalyst and their integration into a fully heterogeneous multi-enzyme system.

The manufacturing of aminoalcohols is an appealing transformation for industrial biocatalysis due to their high value as building blocks in synthetic chemistry. By co‐immobilizing dehydrogenases, ω‐transaminases and oxidases on porous carriers, we fabricated and characterized a multi‐functional heterogeneous biocatalyst that was further applied to the cell‐free biosynthesis of amines and aminoalcohols from alcohols and diols, respectively. This immobilized cascade integrates both redox cofactor and amine donor recycling systems into the amino alcohol biosynthesis. Spatial co‐localization of the multi‐enzyme system significantly increases the cofactor recycling efficiency, the system productivity and the product conversion. Using this multi‐functional heterogeneous biocatalyst, we achieved 80 % conversion of alcohols into amines and accessed a palette of up to 5 aminoalcohols starting from their corresponding diols in one‐pot. The results herein presented contribute to understanding the effects of the confinement of co‐immobilized enzymes on the cascade processes.

The activity orchestration of an unprecedented cell‐free enzyme system with self‐sufficient cofactor recycling enables the stepwise transformation of aliphatic diols into ω‐hydroxy acids at the expense of molecular oxygen as electron acceptor. The efficiency of the biosynthetic route was maximized when two compatible alcohol dehydrogenases were selected as specialist biocatalysts for each one of the oxidative steps required for the oxidative lactonization of diols. The cell‐free system reached up to 100 % conversion using 100 mM of linear C5 diols and performed the desymmetrization of prochiral branched diols into the corresponding ω‐hydroxy acids with an exquisite enantioselectivity (ee>99 %). Green metrics demonstrate superior sustainability of this system compared to traditional metal catalysts and even to whole cells for the synthesis of 5‐hydroxypetanoic acid. Finally, the cell‐free system was assembled into a consortium of heterogeneous biocatalysts that allowed the enzyme reutilization. This cascade illustrates the potential of systems biocatalysis to access new heterofunctional molecules such as ω‐hydroxy acids.

Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger a substrate channeling effect, allowing for efficient cofactor reuse with industrial potential. However, precise nanometric organization of enzymes challenges the design of scaffolds. In this study, we create a nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding for biocatalysis. We genetically fuse TRAP domains and program them to selectively and orthogonally recognize peptide-tags fused to enzymes, which upon binding form spatially organized metabolomes. In addition, the scaffold encodes binding sites to selectively and reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, the catalytic efficiency. This concept is demonstrated for the biosynthesis of amino acids and amines using up to three enzymes. Scaffolded multi-enzyme systems present up to 5-fold higher specific productivity than the non-scaffolded ones. In-depth analysis suggests that channeling of NADH cofactor between the assembled enzymes enhances the overall cascade throughput and the product yield. Moreover, we immobilize this biomolecular scaffold on solid supports, creating reusable heterogeneous multi-functional biocatalysts for consecutive operational batch cycles. Our results demonstrate the potential of TRAP-scaffolding systems as spatial-organizing tools to increase the efficiency of cell-free biosynthetic pathways.